Expression of Hepatitis C Virus Ns5b Protein: Characterization of Its Rna Polymerase Activity and Rna Binding

Abstract

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is considered to possess RNA-dependent RNA polymerase (RdRp) activity and to play an essential role for the viral replication. In this study, we expressed the NS5B protein of 65 kd by a recombinant baculovirus. With the highly purified NS5B protein, we established an in vitro system for RdRp activity by using poly(A) as a template and a 15-mer oligo(U) (oligo(U) 15 ) as a primer. Optimal conditions of temperature and pH for primer-dependent polymerase activity of the NS5B were 32ЊC and pH 8. Hepatitis C virus (HCV) is the major etiologic agent of both posttransfusion and sporadic non-A, non-B hepatitis throughout the world. 1-3 HCV infection often leads to persistence, resulting in chronic hepatitis, and develops into hepatocellular carcinoma. Although the incidence of infection with HCV was drastically decreased by introduction of an efficient blood screening system, 1% to 2% of the worldwide population are already infected with HCV. Furthermore, seroepidemiological studies indicated that there is a clear correlation between infection with HCV and the development of hepatocellular carcinoma. It is therefore very important to develop anti-HCV agents with different antiviral mechanisms for the treatment of liver diseases associated with HCV infection. A number of other drugs with a broad spectrum of antiviral activity have been given to patients with hepatitis C. 4 To date, however, few convincing positive results have been obtained.HCV is a positive-stranded RNA virus with the genome size of approximately 9,400 bases and contains a large open reading frame encoding a precursor polyprotein of about 3,000 amino acids. [5][6][7][8] From their deduced amino acid sequence, HCV was shown to be related to pestiviruses and distantly related to flaviviruses. [8][9][10][11] Recent studies revealed that this polyprotein was cleaved into at least 9 mature proteins by host cellular signalases and viral coded proteinases. 12,13 However, because of the lack of an efficient in vitro cell culture system to grow the virus, little is known about the mechanisms of the replication of HCV. Synthesis of both replicative intermediate minus-strand HCV RNA and progeny positive-strand HCV RNA is thought to occur via an RNA-dependent RNA polymerase (RdRp) reaction. The nonstructural 5B (NS5B) protein of 65 kd is located at the C-terminal region of the HCV polyprotein and shares its sequence with those of other proteins of animal and plant viruses and bacteriophages possessing RdRp activity. 14,15

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

“…This result suggests that Cterminal region of NS5B might play a role in the initiation of RNA synthesis from 3'-end of HCV genome. To date, HCV NS5B polymerase has been shown to initiate RNA synthesis by snap-back or de novo mechanisms using a natural HCV RNA template (Behrens et al, 1996;Lohmann et al, 1997;Yamashita et al, 1998;Ishii et al, 1999;Oh et al, 2000;Sun et al, 2000;Ranjith-Kumar et al, 2001;Reigadas et al, 2001;Kashiwagi et al, 2002;Kim et al, 2002;Pellerin et al, 2002). Our results shown with several HCV RNA templates using TNTase-free NS5B are in agreement with the previous result obtained with E. coli-expressed NS5B (Oh et al, 1999), indicating that HCV NS5B proteins initiate RNA synthesis in a primer-independent manner.…”

“…RNA product size increased steadily up to 120 min when the product reached the template size ( Figure 3A, lane 6). The kinetics of RNA synthesis using a full-length HCV RNA template over 360 min timecourse indicated that RNA synthesis is not initiated from the 3'-snap-back structure, in contrast to previous reports using TNTase-associated HCV NS5B proteins purified from insect cells (Behrens et al, 1996;Lohmann et al, 1997;Ishii et al, 1999). Next, we investigated whether HCV NS5B can perform cyclic replication without help of cellular and/or viral factors; synthesis of complementary RNA using the input RNA template and subsequent use of the RNA products for the synthesis of original input RNA template.…”

“…For example, C-terminal 21 amino acid truncated NS5B fused with glutathione S transferase (GST) generated two RNA products, one longer and one shorter than the template X-RNA (Yamashita et al, 1998). In addition, insect cell-expressed full-length NS5B generated approximately template-size labeled X-RNA by action of TNTase associated with NS5B (Ishii et al, 1999). Other previous studies have also shown that HCV NS5B proteins expressed in insect cells are associated with host TNTase (Behrens et al, 1996;Lohmann et al, 1997).…”

Baseline dasabuvir resistance in Hepatitis C virus from the genotypes 1, 2 and 3 and modeling of the NS5B-dasabuvir complex by thein silicoapproach

“…However, these residues are more conserved as previously shown and D318 is one of the residues constituting the NS5B polymerase’s active site [42]. Moreover, the importance of the residue position 291 was demonstrated by Ishii et al which found that by introducing the mutations N291A and T287A in recombinant NS5B completely abolished the polymerase activity [43]. In our study, we found a high prevalence of variants C445F, A553V and S556G in both GTs 2b and 3a.…”

Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture

“…It is also close to Arg-222, which is involved in NTP trafficking (49). aa 288 is located in motif B, near the catalytic center of NS5B, and is also near aa 286, 287, and 291, of which mutations were reported to result in defective RdRP activity (11,13,46). Mutations at aa 287 and 291 might also be involved in RNA binding (11).…”